1. Field of the Invention
This invention relates to analytical chemistry and in particular to an improved multilayer analytical element for analysis of body fluids for specific components, and more specifically to an element useful in the analysis of blood serum for blood urea nitrogen (BUN) content.
2. State of the Prior Art
In recent years, a number of automated systems for carrying out chemical analyses of fluid samples have been developed; and these have proven particularly advantageous for use in clinical laboratories, especially in the quantitative analysis of blood. Systems based on continuous flow analysis in which sample, diluents and test reagents are mixed together and transported through the analyzer are very widely utilized. However, these continuous analyzers, such as, for example, the analyzer illustrated in U.S. Pat. No. 2,797,149, are complex and expensive, require skilled operators, necessitate considerable expenditure of time and effort in repetitive cleaning operations, and do not permit the use of very small quantities of sample, such as are used in microanalytical techniques. In an effort to overcome these disadvantages, automated chemical analyzers have been proposed which utilize a continuous analytical tape on which the sample to be analyzed is deposited and which effect quantitative analysis by means of spectrophotometric measurement of color-forming reactions between constituents of the sample to be analyzed and test reagents applied to or carried by the tape. Analyzers of this type are described in detail in many patents, for example, in British Pat. No. 1,049,364 and in U.S. Pat. Nos. 3,036,893 and 3,526,480.
The analyzers utilizing continuous analytical tapes are inherently much simpler than continuous flow analyzers. However, analytical tape analyzers such as are described in the aforesaid patents suffer from many significant disadvantages which have hindered their commercial development. Thus, where the tape does not incorporate the test reagents within itself and is merely utilized as a means of transporting the sample to be analyzed through the system, as in certain embodiments of British Pat. No. 1,049,364, provision must be made for separate application of sample and test reagents to the tape at the right time and in the right amounts. The use of separate tapes to accomplish various functions, such as sample filtering, and reaction of the sample with the test reagents, as in U.S. Pat. No. 3,036,893 and in certain other embodiments of British Pat. No. 1,049,364, adds greatly to the complexity of the system so that the inherent simplicity of the continuous tape method of analysis is not fully realized. Use of analytical tapes of a complex nature which are difficult and expensive to manufacture, such as are described in U.S. Pat. No. 3,526,480 is, of course, also a serious hindrance to achieving a commercially practical system.
Other "dry" systems to perform quantitative or semi-quantitative determination of blood components have also been devised. These include primarily the incorporation of reagents into bibulous carrier materials such as paper or porous hydrophilic polymeric layers which may or may not also include overcoats of dialyzing materials to filter out undesirable blood components such as red cells. Specifically, in the case of blood urea nitrogen (hereafter BUN), urease is imbibed into a bibulous carrier overcoated with a dialyzing membrane (U.S. Pat. Nos. 3,145,086; 3,249,513; 3,395,082; and Brit. Pat. No. 1,287,785). According to the teachings of these patents, the ammonia released by the reaction of urea with oxygen in the presence of urease initiates a color change in an imbibed pH indicator. However, the usefulness of these systems is adversely affected by the presence of other basic materials which falsely affect the indicator. The dialyzing membrane, if used, is only a macrofilter positioned as the outermost layer. Barrier bands are disclosed as options, but these are used to divide the test strip lengthwise into regions of different sensitivity, and these barriers do not function to permit selective migration into interior layers.
The disclosure of U.S. Pat. No. 3,011,874 indicates that other barrier bands have been considered for BUN test elements heretofore. However, as disclosed in this patent, the barrier is a barrier to all substances, the ammonia being released by a gas release band for atmospheric diffusion to the indicator band in a closed container.
Still other systems have been developed based upon the urease reaction, but these have been largely confined to liquid reactions. Patents representative of this approach include U.S. Pat. Nos. 3,409,508 and 3,485,723. Still other patents which are relevant only to the general background of BUN analysis include U.S. Pat. Nos. 3,718,433; 3,655,516; 3,567,374; 3,511,611, and 3,531,254.
U.S. Pat. No. 3,723,064 provides a layered test element useful in clinical analysis, wherein two reagent layers are separated by intermediate layers. One of these layers may have chemical traps of varying concentration, which are specific to the end product being tested for. Barriers may be provided for the intermediate layers in the form of a plurality of vertically extending barrier compositions such as cellulose acetate to control concentration gradients flowing through the intermediate layer. The barriers do not function to intercept interfering or undesired components as they flow from one reagent layer to the other in their normal direction of flow. Furthermore, they vary in concentration within the sample intercept area of the element.
U.S. Pat. No. 3,901,657, describes a filter paper layer as a physical separation between a reagent-containing layer and an indicator layer. The entire liquid sample and its components are permitted to pass through the filter paper. No suggestion is made that the paper layer should selectively pass only a portion of an applied sample or a decomposition product of the analytical reaction.
A recent approach to the problem of blood analysis is disclosed in commonly-owned U.S. application Ser. No. 538,072, filed Jan. 2, 1975, entitled "Integral Analytical Element," by Edwin P. Przybylowicz et al., now U.S. Pat. No. 3,992,158. In this approach, an element comprising a reagent layer, an isotropically porous spreading layer, and additional layers which may include a dialysis layer, a filtering layer, a reflecting layer, or a combination of any of these is used to generate a detectable change, such as a quantifiable shift in energy absorption or transmission in response to the presence of an analyte in a sample being tested. The reagent layer is in fluid contact with the spreading layer, as are the additional layers, so that the component being analyzed or a precursor thereof or reaction product thereof will reach the reagent layer. The suggested filter or dialysis layer(s) are described as functioning to exclude passage of filterable materials present in the sample, and to pass sample components not trapped in the filter layer. Such filter or dialysis layers are not described as being specifically concerned with preferential passage of a decomposition product produced directly or indirectly by materials contained in a reagent layer positioned ahead of the filter in the normal flow of the fluid under study, to the exclusion of possibly interfering substances.
Enzyme electrodes using semipermeable membranes selectively permeable to a material of choice have been used in conjunction with an enzyme layer and an electrode means for potentiometrically registering a decomposition product produced by the enzyme, such as, ammonia produced from urea in the presence of urease. Such devices are disclosed, for example, in U.S. Pat. No. 3,838,033 and U.S. Pat. No. 3,896,008.